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Metabolic Pathways and Defects in Fructose Metabolism

果糖代谢的代谢途径和缺陷

基本信息

批准号：
7476023
负责人：
Dean R. TOLAN
金额：
$ 5.18万
依托单位：
BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-08-01 至 2009-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7476023
关键词：
African American Aldehyde-Lyases Aldolase B Algorithms Alleles American Animal Model Animals Assimilations Bacteria Binding Biochemical Biochemical Genetics Bioinformatics Biological Assay Blood specimen Clinical Code Complement Complement component C1s Complex Corticosterone Crystallography DNA Microarray Chip DNA Microarray format DNA Sequence DNA Sequence Determination Data Databases Defect Development Diabetes Mellitus Diagnosis Diagnostic Diagnostic Procedure Diet Digestion Disease Drug Design Enzymes Ethnic group Exons Expressed Sequence Tags Family Fasting Fatty Acids Fructokinases Fructose Fructosediphosphate Aldolase GLUT2 gene Gene Expression Gene Targeting Genes Genetic Structures Genetic Transcription Genotype Glucose Goals Health Heart Diseases Hereditary Disease Hereditary fructose intolerance syndrome Hexose Transporter Hispanic Americans Hispanics Human Immunohistochemistry In Situ Hybridization Inborn Genetic Diseases Incubated Individual Ingestion Insulin Invasive Investigation Isoenzymes Ketohexokinase Kidney Knock-in Mouse Knock-out Knockout Mice Knowledge Laboratories Libraries Ligands Liver Messenger RNA Metabolic Metabolic Pathway Metabolism Modeling Molecular Molecular Profiling Mus Mutation Obesity Oligonucleotides Operative Surgical Procedures Pathology Pathway interactions Pattern Personal Satisfaction Pharmaceutical Preparations Phenotype Phosphotransferases Physiological Physiology Play Population Procedures RNA Radioactive Recovery Resolution Reverse Transcriptase Polymerase Chain Reaction Role Screening procedure Site Slice Small Intestines Standards of Weights and Measures Structure Techniques Technology Temperature Testing Therapeutic Therapeutic Agents Tissues Triglycerides Trioses Validation X-Ray Crystallography base cell type chemical genetics combinatorial chemistry design diabetic dimer embryonic stem cell fructose-1,6-diphosphate fructose-1-phosphate fructose-6-phosphate hexokinase high throughput screening molecular modeling mouse model mutant novel oxidation prescription document prescription procedure recombinase research study restriction enzyme segregation size small molecule small molecule libraries sugar tool triokinase vector

项目摘要

Recently, fructose has become an increasing part of the Western diet, even though its ingestion can be harmful. Long-term ingestion has effects on diabetes and obesity. The most drastic and common genetic disorder of fructose metabolism is hereditary fructose intolerance (HFI). Lack of knowledge about sites of fructose metabolism and about genotype-phenotype relationships still exists for this disease and reflects the incomplete understanding of normal fructose metabolism. Answers to two major questions will provide new information. First, other than liver and kidney, what other tissues play a role in fructose metabolism? Second, can small molecules be found that stabilize the major defective enzyme in HFI, that harboring an A149P substitution (AP-aldolase)? The proposed investigations will, 1) define sites for fructose assimilation and utilization using a combination of bioinformatics and molecular approaches, 2) determine a high-resolution structure of AP-aldolase, and use it to find stabilizing small-molecule ligands by both structure-based ligand design (SBLD) and high-throughput screening of chemical libraries, 3) create animal models for HFI using gene-targeting techniques, and 4) identify HFI mutations in the diverse US population, in particular Hispanic, African-American, and other ethnic groups that have not been well characterized, and correlate these findings to any specific phenotypes in these ethnic groups. The large database of expressed sequence tags (dbEST) will be analyzed for overlapping expression profiles of the GLUT5, GLUT2, ketohexokinase, aldolase, hexokinase, and triose kinase in both mouse and humans to predict alternative sites of fructose metabolism. Verification and characterization of these global predictions will be done by quantitative reverse-transcriptase polymerase chain reaction (Q-PCR), RNA in situ hybridization (RISH), and identification of metabolic intermediates during the oxidation of radioactive fructose. For the second hypothesis, a high-resolution structure of AP-aldolase will be determined by macromolecular X-ray crystallography. Screening large libraries of small molecules (produced by combinatorial chemistry) using a thermal-stability assay of AP-aldolase in conjunction with SBLD will identify small molecules that restore enzyme function. Should the gene-targeted animal model mimic the human HFI pathology, the metabolic profiles and sites of fructose metabolism will be determined. Should the animal model be asymptomatic, differences in the ability to metabolize fructose will be compared to previously determined sites and pathways for fructose metabolism in normal mice and humans. Lastly, blood samples from African-American and Hispanic-American HFI subjects will be used for identification of gene defects by direct DNA sequencing, thus offering a reliable non-invasive diagnostic method to these Americans.

最近，果糖已经成为西方饮食中越来越多的一部分，尽管它的摄入可能是有害的。长期摄入对糖尿病和肥胖有影响。最严重和最常见的遗传疾病，果糖代谢是遗传性果糖不耐受（HFI）。缺乏有关果糖位点的知识代谢和关于基因型-表型关系仍然存在这种疾病，并反映了不完全的了解正常的果糖代谢。两个主要问题的答案将提供新的信息.首先，除了肝脏和肾脏，还有哪些组织在果糖代谢中发挥作用？第二、能否找到小分子来稳定HFI中的主要缺陷酶，即携带A149 P 取代（AP-醛缩酶）？拟议的调查将：1）确定果糖同化的地点，利用生物信息学和分子方法的组合，2）确定高分辨率 AP-醛缩酶结构，并利用其通过基于结构的配体两者来寻找稳定的小分子配体设计（SBLD）和化学库的高通量筛选，3）使用创建HFI的动物模型基因靶向技术，以及4）鉴定不同美国人群中的HFI突变，特别是西班牙裔，非洲裔美国人和其他尚未得到很好表征的种族群体，并将这些发现与这些种族群体中的任何特定表型。表达序列标签（dbEST）的大型数据库将在分析GLUT 5、GLUT 2、己酮糖激酶、醛缩酶、己糖激酶和丙糖激酶来预测果糖代谢的替代位点。核查和这些全局预测的表征将通过定量逆转录酶聚合酶链反应（Q-PCR），RNA原位杂交（RISH）和代谢中间体的鉴定放射性果糖的氧化。对于第二个假设，AP-醛缩酶的高分辨率结构将是通过大分子X射线晶体学测定。筛选小分子的大文库（产生的通过组合化学）使用AP-醛缩酶的热稳定性测定结合SBLD将鉴定恢复酶功能的小分子。基因靶向动物模型是否应该模仿人类将确定HFI病理学、代谢谱和果糖代谢部位。如果动物模型无症状，代谢果糖能力的差异将与以前相比，在正常小鼠和人类中确定果糖代谢的位点和途径。最后，血液样本来自非洲裔美国人和西班牙裔美国人的HFI受试者将用于通过以下方式鉴定基因缺陷：直接DNA测序，从而为这些美国人提供可靠的非侵入性诊断方法。